Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies.

The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy-CD19, CD33, and CD123-to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further.

Magnetic Nanoprobes for Spatio-Mechanical Manipulation in Single Cells.

Magnetic nanoparticles (MNPs) are widely known as valuable agents for biomedical applications. Recently, MNPs were further suggested to be used for a remote and non-invasive manipulation, where their spatial redistribution or force response in a magnetic field provides a fine-tunable stimulus to a cell. Here, we investigated the properties of two different MNPs and assessed their suitability for spatio-mechanical manipulations: semisynthetic magnetoferritin nanoparticles and fully synthetic 'nanoflower'-shaped iron oxide nanoparticles. As well as confirming their monodispersity in terms of structure, surface potential, and magnetic response, we monitored the MNP performance in a living cell environment using fluorescence microscopy and asserted their biocompatibility. We then demonstrated facilitated spatial redistribution of magnetoferritin compared to 'nanoflower'-NPs after microinjection, and a higher magnetic force response of these NPs compared to magnetoferritin inside a cell. Our remote manipulation assays present these tailored magnetic materials as suitable agents for applications in magnetogenetics, biomedicine, or nanomaterial research.

Nanoscale FasL Organization on DNA Origami to Decipher Apoptosis Signal Activation in Cells.

Cell signaling is initiated by characteristic protein patterns in the plasma membrane, but tools to decipher their molecular organization and activation are hitherto lacking. Among the well-known signaling pattern is the death inducing signaling complex with a predicted hexagonal receptor architecture. To probe this architecture, DNA origami-based nanoagents with nanometer precise arrangements of the death receptor ligand FasL are introduced and presented to cells. Mimicking different receptor geometries, these nanoagents act as signaling platforms inducing fastest time-to-death kinetics for hexagonal FasL arrangements with 10 nm inter-molecular spacing. Compared to naturally occurring soluble FasL, this trigger is faster and 100× more efficient. Nanoagents with different spacing, lower FasL number or higher coupling flexibility impede signaling. The results present DNA origami as versatile signaling scaffolds exhibiting unprecedented control over molecular number and geometry. They define molecular benchmarks in apoptosis signal initiation and constitute a new strategy to drive particular cell responses.

CD44v6-targeted CAR T-cells specifically eliminate CD44 isoform 6 expressing head/neck squamous cell carcinoma cells.

Immune checkpoint blockade can cause regression of recurrent and/or refractory head and neck squamous cell carcinoma (HNSCC). As a second type of immunotherapy, adoptive cellular therapy with genetically modified patient's T-cells redirected against the autologous malignant cells by expressing chimeric antigen receptors (CARs) recognizing tumor-associated antigens has been established as highly efficient personalized treatment for hematological malignancies. In solid cancers however, the application of these genetically modified immune effector cells still lacks equal response rates. CD44v6 is an isoform of the hyaluronic receptor CD44 that is almost exclusively expressed at high levels on solid cancers and has been associated with tumorigenesis, tumor cell invasion and metastasis. Here, we established a highly specific CAR against CD44v6 on HNSCC cells that can be expressed on normal T-cells with lentiviral vectors. Using primary human HNSCC cells in combination with CRISPR/Cas9 and overexpression approaches allowed us to confirm the high specificity of our CAR construct for the tumor-associated CD44v6 as target antigen and to demonstrate a direct correlation between CD44v6 expression levels and cytotoxicity of the CAR T-cells. Importantly, the design of our clinically applicable lentiviral vector facilitates to co-express a second transgene for in vivo control of CAR T-cells, if undesired side-effects or toxicities occur.

Single-molecule analysis of dynamics and interactions of the SecYEG translocon.

Protein translocation and insertion into the bacterial cytoplasmic membrane are the essential processes mediated by the Sec machinery. The core machinery is composed of the membrane-embedded translocon SecYEG that interacts with the secretion-dedicated ATPase SecA and translating ribosomes. Despite the simplicity and the available structural insights on the system, diverse molecular mechanisms and functional dynamics have been proposed. Here, we employ total internal reflection fluorescence microscopy to study the oligomeric state and diffusion of SecYEG translocons in supported lipid bilayers at the single-molecule level. Silane-based coating ensured the mobility of lipids and reconstituted translocons within the bilayer. Brightness analysis suggested that approx. 70% of the translocons were monomeric. The translocons remained in a monomeric form upon ribosome binding, but partial oligomerization occurred in the presence of nucleotide-free SecA. Individual trajectories of SecYEG in the lipid bilayer revealed dynamic heterogeneity of diffusion, as translocons commonly switched between slow and fast mobility modes with corresponding diffusion coefficients of 0.03 and 0.7 µm2 ·s-1 . Interactions with SecA ATPase had a minor effect on the lateral mobility, while bound ribosome:nascent chain complexes substantially hindered the diffusion of single translocons. Notably, the mobility of the translocon:ribosome complexes was not affected by the solvent viscosity or macromolecular crowding modulated by Ficoll PM 70, so it was largely determined by interactions within the lipid bilayer and at the interface. We suggest that the complex mobility of SecYEG arises from the conformational dynamics of the translocon and protein:lipid interactions.

Fluorescence Correlation Spectroscopy Reveals Interaction of Some Microdomain-Associated Lipids with Cellular Focal Adhesion Sites.

Cells adhere to the extracellular matrix at distinct anchoring points, mostly focal adhesions. These are rich in immobile transmembrane- and cytoskeletal-associated proteins, some of which are known to interact with lipids of the plasma membrane. To investigate their effect on lipid mobility and molecular interactions, fluorescently labeled lipids were incorporated into the plasma membranes of primary myofibroblasts using fusogenic liposomes. With fluorescence correlation spectroscopy, we tested mobilities of labeled microdomain-associated lipids such as sphingomyelin (SM), ganglioside (GM1), and cholesterol as well as of a microdomain-excluded phospholipid (PC) and a lipid-like molecule (DiIC18(7)) in focal adhesions (FAs) and in neighboring non-adherent membrane areas. We found significantly slower diffusion of SM and GM1 inside FAs but no effect on cholesterol, PC, and DiIC18(7). These data were compared to the molecular behavior in Lo/Ld-phase separated giant unilamellar vesicles, which served as a model system for microdomain containing lipid membranes. In contrast to the model system, lipid mobility changes in FAs were molecularly selective, and no particle enrichment occurred. Our findings suggest that lipid behavior in FAs cannot be described by Lo/Ld-phase separation. The observed slow-down of some molecules in FAs is potentially due to transient binding between lipids and some molecular constituent(s).

Biomimetic niches reveal the minimal cues to trigger apical lumen formation in single hepatocytes.

The symmetry breaking of protein distribution and cytoskeleton organization is an essential aspect for the development of apicobasal polarity. In embryonic cells this process is largely cell autonomous, while differentiated epithelial cells collectively polarize during epithelium formation. Here, we demonstrate that the de novo polarization of mature hepatocytes does not require the synchronized development of apical poles on neighbouring cells. De novo polarization at the single-cell level by mere contact with the extracellular matrix and immobilized cadherin defining a polarizing axis. The creation of these single-cell liver hemi-canaliculi allows unprecedented imaging resolution and control and over the lumenogenesis process. We show that the density and localization of cadherins along the initial cell-cell contact act as key triggers of the reorganization from lateral to apical actin cortex. The minimal cues necessary to trigger the polarization of hepatocytes enable them to develop asymmetric lumens with ectopic epithelial cells originating from the kidney, breast or colon.

3D Cellular Architecture Modulates Tyrosine Kinase Activity, Thereby Switching CD95-Mediated Apoptosis to Survival.

The death receptor CD95 is expressed in every cancer cell, thus providing a promising tool to target cancer. Activation of CD95 can, however, lead to apoptosis or proliferation. Yet the molecular determinants of CD95's mode of action remain unclear. Here, we identify an optimal distance between CD95Ligand molecules that enables specific clustering of receptor-ligand pairs, leading to efficient CD95 activation. Surprisingly, efficient CD95 activation leads to apoptosis in cancer cells in vitro and increased tumor growth in vivo. We show that allowing a 3D aggregation of cancer cells in vitro switches the apoptotic response to proliferation. Indeed, we demonstrate that the absence or presence of cell-cell contacts dictates the cell response to CD95. Cell contacts increase global levels of phosphorylated tyrosines, including CD95's tyrosine. A tyrosine-to-alanine CD95 mutant blocks proliferation in cells in contact. Our study sheds light into the regulatory mechanism of CD95 activation that can be further explored for anti-cancer therapies.

Zwitterionic polymer ligands: an ideal surface coating to totally suppress protein-nanoparticle corona formation?

In the last few years, zwitterionic polymers have been developed as antifouling surface coatings. However, their ability to completely suppress protein adsorption at the surface of nanoparticles in complex biological media remains undemonstrated. Here we investigate the formation of hard (irreversible) and soft (reversible) protein corona around model nanoparticles (NPs) coated with sulfobetaine (SB), phosphorylcholine (PC) and carboxybetaine (CB) polymer ligands in model albumin solutions and in whole serum. We show for the first time a complete absence of protein corona around SB-coated NPs, while PC- and CB-coated NPs undergo reversible adsorption or partial aggregation. These dramatic differences cannot be described by naïve hard/soft acid/base electrostatic interactions. Single NP tracking in the cytoplasm of live cells corroborate these in vitro observations. Finally, while modification of SB polymers with additional charged groups lead to consequent protein adsorption, addition of small neutral targeting moieties preserves antifouling and enable efficient intracellular targeting.

Optical Magnetometry of Single Biocompatible Micromagnets for Quantitative Magnetogenetic and Magnetomechanical Assays.

The mechanical manipulation of magnetic nanoparticles is a powerful approach to probing and actuating biological processes in living systems. Implementing this technique in high-throughput assays can be achieved using biocompatible micromagnet arrays. However, the magnetic properties of these arrays are usually indirectly inferred from simulations or Stokes drag measurements, leaving unresolved questions about the actual profile of the magnetic fields at the micrometer scale and the exact magnetic forces that are applied. Here, we exploit the magnetic field sensitivity of nitrogen-vacancy color centers in diamond to map the 3D stray magnetic field produced by a single soft ferromagnetic microstructure. By combining this wide-field optical magnetometry technique with magneto-optic Kerr effect microscopy, we fully analyze the properties of the micromagnets, including their magnetization saturation and their size-dependent magnetic susceptibility. We further show that the high magnetic field gradients produced by the micromagnets, greater than 104 T·m-1 under an applied magnetic field of about 100 mT, enables the manipulation of magnetic nanoparticles smaller than 10 nm inside living cells. This work paves the way for quantitative and parallelized experiments in magnetogenetics and magnetomechanics in cell biology.

Simple Physical Model Unravels Influences of Chemokine on Shape Deformation and Migration of Human Hematopoietic Stem Cells.

We studied the dynamic behavior of human hematopoietic stem cells (HSC) on the in vitro model of bone marrow surfaces in the absence and presence of chemokine (SDF1α). The deformation and migration of cells were investigated by varying the chemokine concentration and surface density of ligand molecules. Since HSC used in this study were primary cells extracted from the human umbilical cord blood, it is not possible to introduce molecular reporter systems before or during the live cell imaging. To account for the experimental observations, we propose a simple and general theoretical model for cell crawling. In contrast to other theoretical models reported previously, our model focuses on the nonlinear coupling between shape deformation and translational motion and is free from any molecular-level process. Therefore, it is ideally suited for the comparison with our experimental results. We have demonstrated that the results in the absence of SDF1α were well recapitulated by the linear model, while the nonlinear model is necessary to reproduce the elongated migration observed in the presence of SDF1α. The combination of the simple theoretical model and the label-free, live cell observations of human primary cells opens a large potential to numerically identify the differential effects of extrinsic factors such as chemokines, growth factors, and clinical drugs on dynamic phenotypes of primary cells.

Author Correction: Dynamic cellular phenotyping defines specific mobilization mechanisms of human hematopoietic stem and progenitor cells induced by SDF1α versus synthetic agents.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Dynamic cellular phynotyping defines specific mobilization mechanisms of human hematopoietic stem and progenitor cells induced by SDF1α versus synthetic agents.

Efficient mobilization of hematopoietic stem and progenitor cells (HSPC) is one of the most crucial issues for harvesting an adequate amount of peripheral HSPC for successful clinical transplantation. Applying well-defined surrogate models for the bone marrow niche, live cell imaging techniques, and novel tools in statistical physics, we have quantified the functionality of two mobilization agents that have been applied in the clinic, NOX-A12 and AMD3100 (plerixafor), as compared to a naturally occurring chemokine in the bone marrow, SDF1α. We found that NOX-A12, an L-enantiomeric RNA oligonucleotide to SDF1, significantly reduced the adhesion of HSPC to the niche surface mediated via the CXCR4-SDF1α axis, and stretched the migration trajectories of the HSPC. We found that the stretching of trajectories by NOX-A12 was more prominent than that by SDF1α. In contrast, plerixafor exhibited no detectable interference with adhesion and migration. We also found that the deformation of HSPC induced by SDF1α or plerixafor was also drastically suppressed in the presence of NOX-A12. This novel technology of quantitative assessment of "dynamic phenotypes" by physical tools has therefore enabled us to define different mechanisms of function for various extrinsic factors compared to naturally occurring chemokines.

Magnetic control of cellular processes using biofunctional nanoparticles.

Remote control of cellular functions is a key challenge in biomedical research. Only a few tools are currently capable of manipulating cellular events at distance, at spatial and temporal scales matching their naturally active range. A promising approach, often referred to as 'magnetogenetics', is based on the use of magnetic fields, in conjunction with targeted biofunctional magnetic nanoparticles. By triggering molecular stimuli via mechanical, thermal or biochemical perturbations, magnetic actuation constitutes a highly versatile tool with numerous applications in fundamental research as well as exciting prospects in nano- and regenerative medicine. Here, we highlight recent studies, comment on the advancement of magnetic manipulation, and discuss remaining challenges.

Engineered Ferritin for Magnetogenetic Manipulation of Proteins and Organelles Inside Living Cells.

Magnetogenetics is emerging as a novel approach for remote-controlled manipulation of cellular functions in tissues and organisms with high spatial and temporal resolution. A critical, still challenging issue for these techniques is to conjugate target proteins with magnetic probes that can satisfy multiple colloidal and biofunctional constraints. Here, semisynthetic magnetic nanoparticles are tailored based on human ferritin coupled to monomeric enhanced green fluorescent protein (mEGFP) for magnetic manipulation of proteins inside living cells. This study demonstrates efficient delivery, intracellular stealth properties, and rapid subcellular targeting of those magnetic nanoparticles via GFP-nanobody interactions. By means of magnetic field gradients, rapid spatial reorganization in the cytosol of proteins captured to the nanoparticle surface is achieved. Moreover, exploiting efficient nanoparticle targeting to intracellular membranes, remote-controlled arrest of mitochondrial dynamics using magnetic fields is demonstrated. The studies establish subcellular control of proteins and organelles with unprecedented spatial and temporal resolution, thus opening new prospects for magnetogenetic applications in fundamental cell biology and nanomedicine.

Correction: Magnetic control of cellular processes using biofunctional nanoparticles.

[This corrects the article DOI: 10.1039/C7SC01462G.].

Nanometric thermal fluctuations of weakly confined biomembranes measured with microsecond time-resolution.

We probe the bending fluctuations of bio-membranes using highly deflated giant unilamellar vesicles (GUVs) bound to a substrate by a weak potential arising from generic interactions. The substrate is either homogeneous, with GUVs bound only by the weak potential, or is chemically functionalized with a micro-pattern of very strong specific binders. In both cases, the weakly adhered membrane is seen to be confined at a well-defined distance above the surface while it continues to fluctuate strongly. We quantify the fluctuations of the weakly confined membrane at the substrate proximal surface as well as of the free membrane at the distal surface of the same GUV. This strategy enables us to probe in detail the damping of fluctuations in the presence of the substrate, and to independently measure the membrane tension and the strength of the generic interaction potential. Measurements were done using two complementary techniques - dynamic optical displacement spectroscopy (DODS, resolution: 20 nm, 10 µs), and dual wavelength reflection interference contrast microscopy (DW-RICM, resolution: 4 nm, 50 ms). After accounting for the spatio-temporal resolution of the techniques, an excellent agreement between the two measurements was obtained. For both weakly confined systems we explore in detail the link between fluctuations on the one hand and membrane tension and the interaction potential on the other hand.

Fine Adjustment of Interfacial Potential between pH-Responsive Hydrogels and Cell-Sized Particles.

We quantitatively determined interfacial potentials between cell-sized particles and stimulus-responsive hydrogels using a microinterferometer. The hydrogel is based on physically interconnected ABA triblock copolymer micelles comprising an inner biocompatible PMPC block and two outer pH-responsive PDPA blocks. The out-of-plane temporal fluctuation in the position of the cell-sized particles was calculated from changes in the interference pattern measured by Reflection Interference Contrast Microscopy (RICM), thus yielding the particle-substrate interaction potential V (Δh). Measurements in pH buffers ranging from 7.0 to 7.8 resulted in a systematic reduction in height of the potential minima ⟨Δh⟩ and a concomitant increase in the potential curvature V″ (Δh). The experimental data were analyzed by applying the modified Ross and Pincus model for polyelectrolytes, while accounting for gravitation, lubrication and van der Waals interactions. Elastic moduli calculated from V″ (Δh) were in good agreement with those measured by Atomic Force Microscopy. The ability to fine-tune both the gel elasticity and the interfacial potential at around physiological pH makes such triblock copolymer hydrogels a promising biocompatible substrate for dynamic switching of cell-material interactions.

Quantifying adhesion mechanisms and dynamics of human hematopoietic stem and progenitor cells.

Using planar lipid membranes with precisely defined concentrations of specific ligands, we have determined the binding strength between human hematopoietic stem cells (HSC) and the bone marrow niche. The relative significance of HSC adhesion to the surrogate niche models via SDF1α-CXCR4 or N-cadherin axes was quantified by (a) the fraction of adherent cells, (b) the area of tight adhesion, and (c) the critical pressure for cell detachment. We have demonstrated that the binding of HSC to the niche model is a cooperative process, and the adhesion mediated by the CXCR4- SDF1α axis is stronger than that by homophilic N-cadherin binding. The statistical image analysis of stochastic morphological dynamics unraveled that HSC dissipated energy by undergoing oscillatory deformation. The combination of an in vitro niche model and novel physical tools has enabled us to quantitatively determine the relative significance of binding mechanisms between normal HSC versus leukemia blasts to the bone marrow niche.